Augmenting hematoma-scavenging capacity of innate immune cells by CDNF reduces brain injury and promotes functional recovery after intracerebral hemorrhage

During intracerebral hemorrhage (ICH), hematoma formation at the site of blood vessel damage results in local mechanical injury. Subsequently, erythrocytes lyse to release hemoglobin and heme, which act as neurotoxins and induce inflammation and secondary brain injury, resulting in severe neurological deficits. Accelerating hematoma resorption and mitigating hematoma-induced brain edema by modulating immune cells has potential as a novel therapeutic strategy for functional recovery after ICH. Here, we show that intracerebroventricular administration of recombinant human cerebral dopamine neurotrophic factor (rhCDNF) accelerates hemorrhagic lesion resolution, reduces peri-focal edema, and improves neurological outcomes in an animal model of collagenase-induced ICH. We demonstrate that CDNF acts on microglia/macrophages in the hemorrhagic striatum by promoting scavenger receptor expression, enhancing erythrophagocytosis and increasing anti-inflammatory mediators while suppressing the production of pro-inflammatory cytokines. Administration of rhCDNF results in upregulation of the Nrf2-HO-1 pathway, but alleviation of oxidative stress and unfolded protein responses in the perihematomal area. Finally, we demonstrate that intravenous delivery of rhCDNF has beneficial effects in an animal model of ICH and that systemic application promotes scavenging by the brain’s myeloid cells for the treatment of ICH.


RNA sequencing from the hemorrhagic striatum of Wt and Cdnf -/mice
Mice were sacrificed at 6 hours post-ICH induction and perfused with 0.9% saline solution before collecting tissue (n=3 for Wt, n=3 for Cdnf -/-). RNA was extracted from punch samples of tissue taken from the hemorrhagic striatum with 1 mm thick sections from positions A/P -0.9 to +0.1. RNA was extracted with Trizol reagent and treated with DNase (#1906, Ambion). RNA purity and quantification were checked using SimpliNano™ -Biochrom Spectrophotometers (Biochrom, MA, USA). RNA degradation and integrity were monitored by Qsep 100 DNA/RNA Analyzer (BiOptic Inc., Taiwan). A total amount of 1 μl RNA per sample was used as input material for RNA sample preparations. Following the manufacturer's recommendations, sequencing libraries were generated using the KAPA mRNA HyperPrep Kit (KAPA Biosystems, Roche, Basel, Switzerland), and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using magnetic oligo-dT beads. Captured mRNA was fragmented by incubating it at 94 °C in the presence of magnesium in KAPA Fragment, Prime, and Elute Buffer (1x). First-strand cDNA was synthesized using random hexamer priming. Combined second-strand synthesis and A-tailing, which converts the cDNA: RNA hybrid into double-stranded cDNA (dscDNA), was used to incorporate dUTP into the second cDNA strand, and then dAMP was added to the 3' ends of the resulting dscDNA. dsDNA adapters with 3'dTMP overhangs were ligated to library insert fragments to generate library fragments carrying the adapters. To select cDNA fragments of 300~400bp in length, fragments were purified with the KAPA Pure Beads system (KAPA Biosystems, Roche, Basel, Switzerland). The library carrying appropriate adapter sequences at both ends was amplified using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Roche, Basel, Switzerland) and library amplification primers. The strand marked with dUTP was not amplified, allowing strand-specific sequencing. Lastly, PCR products were purified using the KAPA Pure Beads system, and the library quality was assessed using the Qsep 100 DNA/RNA Analyzer (BiOptic Inc., Taiwan). The RNA-seq data (Supplement data 2) has been deposited in the RNA-Seq database at Biotools-rat-RNA pre library under accession number PRJNA745332.

Oxidative damage of proteins by Oxyblot
To determine the amount of carbonylated proteins, we used the OxyBlot Protein Oxidation Detection Kit (Chemicon International, S7150), as described by the manufacturer's instructions. Tissues or cells were lysed in RIPA lysis buffer containing a protease inhibitor cocktail (Roche). 5 mg of tissue/cell lysate in 10 ml lysis buffer with 12% SDS was added to 10 µl of 10 mM 2,4-dinitrophenylhydrazine (DNPH) solution for 15 minutes at RT for derivatizing the sample. As a control, 10 µl of a duplicate sample was added to 10 µl of a control solution not containing DNPH. Both samples were subsequently incubated with 7.5 µl of a neutralization solution to stop the derivatization reaction.
Proteins were separated by 12% SDS-PAGE and transferred to PVDF membranes. DNP-containing proteins were immunostained using rabbit anti-DNP antiserum (1:2000) and goat anti-rabbit IgG conjugated to horseradish peroxidase (HRP) (1:2000). Blots were visualized by an enhanced chemiluminescence (ECL) system. The results were quantified by Image J software.

Assessment of cytokines
Ipsilateral striatal tissues were used to evaluate the cytokine expression by an enzyme-linked immunosorbent assay (ELISA) kits for TNFα (DY510) were normalized to 50 μg of protein.

Evaluation of physiological parameters
Under urethane (1.0 g/kg body weight, i.p., Sigma-Aldrich) anesthesia, a femoral artery was cannulated with a PE-50 polyethylene tube for fluid supplementation and monitoring of arterial blood pressure and blood gas. Arterial blood pressure and heart rate were recorded through an amplifier (MP36, BIOPAC system, CA, USA) and stored in a PC. Body temperature (rectal temperature) was automatically maintained at 37.5 ± 0.5°C by a rectal temperature sensor and a heating pad (CMA-150, Sweden). Physiological parameters including PaO2, sodium, potassium, glucose, lactate, and hemoglobin) were measured 10 minutes pre-(Pre-op) and 3, 6, and 24 hours post-ICH.

Statistical Analysis
Values are presented as mean ± S.E.M. Unpaired t-test, and one-or two-way analysis of variance (ANOVA) with post hoc Bonferroni tests were used for statistical analysis. A statistically significant difference was defined as p<0.05.